GSM6021847: OIR_control_P12_4; Mus musculus; RNA-Seq
Dissected retinae were stored in RNAlater (Invitrogen).  RNAlater-preserved tissues were disrupted and homogenized in CK14 tubes (VWR, 10144-496) with RLT buffer using a Precellys Evolution homogenizer (Bertin Technologies). Total RNA was then extracted using the MagMAX-96 total RNA isolation kit (Thermo Fisher Scientific, AM1830) including a DNase digestion prior to final elution. Total RNA input of 100 ng was used for library construction with the TruSeq Stranded mRNA LT kit—Set A (Illumina, RS-122-2101), together with the SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, 18064014), according to the manufacturer's protocol. The quality of mRNA libraries was assessed for adapter and heterodimer presence using the DNA 1000 microfluidic chip (Agilent, 5067-1504), while library molarity was measured using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, P11496). All 32 sequencing libraries were then normalized, pooled and spiked in with PhiX Control v3 (Illumina). The library pool was subsequently clustered using the TruSeq SR Cluster Kit v3 (Illumina, GD-401-3001), and sequenced with TruSeq SBS Kit v3 reagents (Illumina, FC-401-3002) on a HiSeq 2000 Sequencing System (Illumina) with single index, single-read reads at 1 x 51 bp length (Read parameters: Rd1: 52, Rd2: 7), reaching an average depth of 24.9 million Pass-Filter reads per sample (13.1% CV).